The role of Endothelial Cell Specific Chemotaxis Regulator (ECSCR) in endothelial cell biology By Ana Raquel Andrade Veríssimo A Thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Immunity and Infection College of Medical and Dental Sciences The University of Birmingham September 2012 i University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Angiogenesis, although a natural process, when deregulated can be associated with many human pathologies, including cancer. The endothelial cells play a crucial role in the growth and remodelling of the vasculature. A novel unique endothelial marker, endothelial cell specific chemotaxis regulator (ECSCR), has been identified and the aim of this project was to characterise its expression patterns and mechanism of action. In HUVECs the interaction with filamin A was confirmed and immunoprecipitation has pulled down several cytoskeletal proteins involved in cell movement, including moesin, that binds to ECSCR. ECSCR loss causes cytoskeleton to be disorganised and overexpression induces filopodia in HEK 293T cells, but not M2 cells, that do not contain filamin A. In zebrafish ECSCR is restricted to the major trunk and head vasculature, whereas in mouse it appears to be present also in the intersegmental vessels. Morpholino mediated knockdown of zebrafish Escr caused disruption of intersegmental vessel sprouting from the trunk vessels. The original phenotype was successfully rescued. A null Ecscr knockout mouse has been produced and preliminary data show that the deletion is not lethal and ECSCR is present in some small vessels, but not all vasculature. Loss of ECSCR may result in respiratory complications. i ACKNOWLEDGEMENTS First and foremost, I would like to express my gratitude to my supervisor, Professor Roy Bicknell. Thank you very much for all your patience, support and advice. It was a great pleasure and a privilege to be a part of your team. I thank Rajeeb, my supervisor for a year, who taught me a great deal about fish and my other fish supervisor, Dr, Ferenc Mueller. Many thanks to all the Bicknell lab members, past and present, effective and extended, by neighbouring effect. Life would not have been the same without the lunchtime daily crosswords. Thank you to my friends, especially Anabela and Vanessa, for all your friendship and support. Finally, I would like to thank my family, especially my parents, for being there, always. ii TABLE OF CONTENTS Abstract ................................................................................................................................ i Acknowledgements ............................................................................................................. ii Table of CONTENTS ............................................................................................................ iii List of figures ...................................................................................................................... vii Abbreviations .................................................................................................................... viii 1 General introduction ........................................................................................................ 1 1.1 The role of endothelial cells in angiogenesis ...................................................... 2 1.2 Developmental and pathological angiogenesis .................................................. 2 1.3 Targeting angiogenesis ....................................................................................... 3 1.4 The identification and characterization of endothelial cell specific chemotaxis regulator (ECSCR) ............................................................................................................ 6 1.5 Cell migration: the fine orchestration between protrusion, retraction and adhesion ........................................................................................................................ 12 1.5.1 Filopodia and lamellipodia at the leading edge of migrating cells ........... 12 1.5.2 The role of cell-matrix adhesions in cell migration ................................... 16 1.6 Cell-cell adhesion .............................................................................................. 19 1.7 Filamins and cytoskeleton ................................................................................. 19 1.8 Angiogenesis assays and models ...................................................................... 27 1.9 Hypothesis ......................................................................................................... 30 1.10 Aim of the study ................................................................................................ 31 2 Materials and Methods ............................................................................................. 33 2.1 Reagents and equipment .................................................................................. 34 2.1.1 Buffers and solutions ................................................................................ 34 2.1.2 Antibodies ................................................................................................. 37 2.1.3 Cloning vectors .......................................................................................... 39 2.1.4 Oligonucleotides ....................................................................................... 40 2.2 Mammalian cell culture and related techniques .............................................. 43 2.2.1 Mammalian cells ....................................................................................... 43 2.2.2 Cell culture ................................................................................................ 43 2.2.3 siRNA knock down of ECSCR in HUVECs ................................................... 45 iii 2.2.4 Polyethylenimine (PEI) transfection of HEK 293T cells ............................. 45 2.2.5 Scratch wound assay ................................................................................. 46 2.2.6 Immunofluorescent staining ..................................................................... 47 2.3 Protein analyses and assays .............................................................................. 47 2.3.1 Cell lysis and protein extraction ................................................................ 47 2.3.2 Immunoprecipitation using an anti-ECSCR intracellular (icd) domain antibody 48 2.3.3 SDS-PAGE .................................................................................................. 48 2.3.4 Gel staining of proteins with Coomassie Brilliant Blue ............................. 48 2.3.5 In-gel digestion of proteins separated on SDS-PAGE gel for mass spectrometry analysis ............................................................................................... 49 2.3.6 Expressing zebrafish Ecscr intracellular domain (icd) fused to MBP ........ 50 2.3.7 GST pull down ........................................................................................... 51 2.3.8 Western Blot ............................................................................................. 51 2.3.9 Yeast-two-hybrid assay ............................................................................. 52 2.3.10 Luciferase complementation assay ........................................................... 54 2.4 Molecular biology methods .............................................................................. 55 2.4.1 Bacterial strains and media ....................................................................... 55 2.4.2 DNA amplification by PCR ......................................................................... 56 2.4.3 DNA digestion using restriction enzymes ................................................. 56 2.4.4 Nucleic acids electrophoresis in agarose gel ............................................ 56 2.4.5 Plasmid ligation and bacterial transformation ......................................... 57 2.4.6 Plasmid preparation and purification ....................................................... 57 2.4.7 Cell lysis and RNA extraction ..................................................................... 58 2.4.8 cDNA synthesis .......................................................................................... 58 2.4.9 Quantitative RT-PCR (qPCR) ...................................................................... 59 2.4.10 Microarray ................................................................................................. 59 2.5 Methods employed for in vivo analyses ........................................................... 60 2.5.1 Species and strains .................................................................................... 60 2.5.2 Zebrafish and mouse whole mount in situ hybridization (WISH) ............. 60 2.5.3 Injection of antisense zebrafish Ecscr morpholino oligonucleotides.......